Levente Szendrei

Heme oxygenase-1 related carbon monoxide production and ventricular fibrillation in isolated ischemic/reperfused mouse myocardium

Heme oxygenase‐1 (HO‐1)‐dependent carbon monoxide (CO) production related to reperfusion‐induced ventricular fibrillation (VF) was studied in HO‐1 wild‐type (+/+), heterozygous (+/−), and homozygous (−/−) isolated ischemic/reperfused mouse heart. In HO‐1 homozygous myocardium, under aerobic conditions, HO‐1 enzyme activity, HO‐1 mRNA, and protein expression were not detected in comparison with aerobically perfused wild‐type and heterozygous myocardium. In wild‐type, HO‐1 hetero‐ and homozygous hearts subjected to 20 min ischemia followed by 2 h of reperfusion, the expression of HO‐1 mRNA, protein, and HO‐1 enzyme activity was detected in various degrees. A reduction in the expression of HO‐1 mRNA, protein, and enzyme activity in fibrillated wild‐type and heterozygous myocardium was observed. In reperfused/nonfibrillated wild‐type and heterozygous hearts, a reduction in HO‐1 mRNA, protein expression, and HO‐1 enzyme activity was not observed, indicating that changes in HO‐1 mRNA, protein, and enzyme activity could be related to the development of VF. These changes were reflected in the HO‐1‐related endogenous CO production measured by gas chromatography. In HO‐1 knockout ischemic/reperfused myocardium, all hearts showed VF, and no detection in HO‐1 mRNA, protein, and enzyme activity was observed. Thus, interventions that are able to increase endogenous CO may prevent the development of VF.

The role of heme oxygenase-related carbon monoxide and ventricular fibrillation in ischemic/reperfused hearts

Reperfusion-induced ventricular fibrillation (VF) and heme oxygenase (HO)-related carbon monoxide (CO) production in isolated ischemic/reperfused rat hearts were studied by gas chromatography. Hearts were subjected to 30 min ischemia followed by 2 h reperfusion, and the expression of HO-1 mRNA (about 4-fold) was observed in ischemic/reperfused-nonfibrillated hearts. In fibrillated hearts, the reduction (about 75%) in HO-1 mRNA expression was detected. These changes in HO-1 mRNA expression were reflected in tissue CO production. Thus, in the absence of VF, CO production was increased about 3.5-fold, while in the presence of VF, CO production was under the detectable level in comparison with the control group. Our results suggest that the stimulation of HO-1 mRNA expression may lead to the prevention of reperfusion VF via an increase in endogenous CO production. To prove this, hearts were treated with 1 microM of N-tert-butyl-alpha-phenylnitrone (PBN) as an inducer of HO-1. PBN treatment resulted in about 20 times increase in HO-1 mRNA expression, and even a higher production rate in endogenous CO. HO protein level and enzyme activity followed the same pattern, as it was observed in HO-1 mRNA expression, in fibrillated and nonfibrillated myocardium. Five mM/l of zinc-protoporphyrin IX (ZnPPIX) significantly blocked HO enzyme activity and increased the incidence of VF, therefore the application of ZnPPIX led to a significant reduction in HO-1 mRNA and protein expression. Our data provide direct evidence of an inverse relationship between the development of reperfusion-induced VF and endogenous CO production. Thus, interventions that are able to increase tissue CO content may prevent the development of reperfusion-induced VF.

Isolation and Analysis of Bioactive Constituents of Sour Cherry (Prunus cerasus) Seed Kernel: An Emerging Functional Food

A plant-based diet reduces the risk for the development of several chronic diseases, such as ischemic heart disease or cancer due to natural compounds found in plants. Numerous cereals, berries, fruits, and vegetables, including sour cherry (Prunus cerasus), which is a favored fruit worldwide, contain biological active components. The antioxidant components of the sour cherry seed kernel have not been investigated until now. The aim of our study was to isolate and analyze the bioactive constituents of sour cherry seed kernel. We separated the oil fraction of the kernel; then the remaining solid fraction was dried, and the oil-free kernel extract was further analyzed. Our results show that sour cherry seed kernel oil contains vegetable oils including unsaturated fatty acids, oleic acids, alpha-tocopherol, tocotrienols, and tocopherol-like components. The components of the solid fraction include various bioactive structures such as polyphenols, flavonoids, vegetable acids, and pro- and anthocyanidins, which could have useful therapeutic effects in the prevention of various vascular diseases.

Postischemic cardiac recovery in heme oxygenase-1 transgenic ischemic/reperfused mouse myocardium

Heme oxygenase‐1 (HO‐1) transgenic mice (Tg) were created using a rat HO‐1 genomic transgene. Transgene expression was detected by RT‐PCR and Western blots in the left ventricle (LV), right ventricle (RV) and septum (S) in mouse hearts, and its function was demonstrated by the elevated HO enzyme activity. Tg and non‐transgenic (NTg) mouse hearts were isolated and subjected to ischemia/reperfusion. Significant post‐ischemic recovery in coronary flow (CF), aortic flow (AF), aortic pressure (AOP) and first derivative of AOP (AOPdp/dt) were detected in the HO‐1 Tg group compared to the NTg values. In HO‐1 Tg hearts treated with 50 μmol/kg of tin protoporphyrin IX (SnPPIX), an HO enzyme inhibitor, abolished the post‐ischemic cardiac recovery. HO‐1 related carbon monoxide (CO) production was detected in NTg, HO‐1 Tg and HO‐1 Tg + SnPPIX treated groups, and a substantial increase in CO production was observed in the HO‐1 Tg hearts subjected to ischemia/reperfusion. Moreover, in ischemia/reperfusion‐induced tissue Na+ and Ca2+ gains were reduced in HO‐1 Tg group in comparison with the NTg and HO‐1 Tg + SnPPIX treated groups; furthermore K+ loss was reduced in the HO‐1 Tg group. The infarct size was markedly reduced from its NTg control value of 37 ± 4% to 20 ± 6% (P < 0.05) in the HO‐1 Tg group, and was increased to 47 ± 5% (P < 0.05) in the HO‐1 knockout (KO) hearts. Parallel to the infarct size reduction, the incidence of total and sustained ventricular fibrillation were also reduced from their NTg control values of 92% and 83% to 25% (P < 0.05) and 8% (P < 0.05) in the HO‐1 Tg group, and were increased to 100% and 100% in HO‐1 KO−/− hearts. Immunohistochemical staining of HO‐1 was intensified in HO‐1 Tg compared to the NTg myocardium. Thus, the HO‐1 Tg mouse model suggests a valuable therapeutic approach in the treatment of ischemic myocardium.

Mitochondrial gene expression and ventricular fibrillation in ischemic/reperfused nondiabetic and diabetic myocardium

We investigated the mitochondrial gene expression related to cardiac function and ventricular fibrillation (VF) in ischemic/reperfused nondiabetic and diabetic myocardium. To identify potentially more specific gene responses we performed subtractive screening, Northern blotting, and reverse transcription-polymerase chain reaction (RT-PCR) of mitochondrial genes expressed after 30 min ischemia followed by 120 min reperfusion in isolated rat hearts that showed VF or did not show VF. Cytochrome oxidase B subunit III (COXBIII) and ATP synthase subunit 6, studied and selected out of 40 mitochondrial genes by subtractive screening, showed an expression after 30 min ischemia (no VF was recorded) in both nondiabetic and diabetic subjects. Upon reperfusion, the down-regulation of these genes was only observed in fibrillated hearts. Such a reduction in signal intensity was not seen in nonfibrillated myocardium. In additional studies, nondiabetic and diabetic hearts, without the ischemia/reperfusion protocol, were subjected to electrical fibrillation, and a significant reduction in COXBIII and ATPS6 mRNA signal intensity was observed indicating that VF contributes to the down-regulation of these genes. Cardiac function (heart rate, coronary flow, aortic flow, left ventricular developed pressure) showed no correlation between the up- and down-regulation of these mitochondrial genes in both nondiabetic and diabetic ischemic/reperfused myocardium. Our data suggest that COXBIII and ATPS6 may play a critical role in arrhythmogenesis, and the stimulation of COXBIII and ATPS6 mRNA expression may prevent the development of VF in both nondiabetic and diabetic ischemic/reperfused myocardium.

Influence of hyperthyroidism on the effect of adenosine transport blockade assessed by a novel method in guinea pig atria.

The aim of the present study was to investigate the effect of hyperthyroidism on the trans-sarcolemmal adenosine (Ado) flux via equilibrative and nitrobenzylthioinosine (NBTI)-sensitive nucleoside transporters (ENT1) in guinea pig atria, by assessing the change in the Ado concentration of the interstitial fluid ([Ado]ISF) under nucleoside transport blockade with NBTI. For the assessment, we applied our novel method, which estimates the change in [Ado]ISF utilizing the altered inotropic response to N6-cyclopentyladenosine (CPA), a relative stable selective agonist of A1 Ado receptors, by providing a relative index, the equivalent concentration of CPA. Our results show an interstitial A do accumulation upon ENT1 blockade, which was more extensive in the hyperthyroid samples (CPA concentrations equieffective with the surplus [Ado]ISF were two to three times higher in hyperthyroid atria than in euthyroid ones, with regard to the negative inotropic effect of CPA and Ado). This suggests an enhanced Ado influx via ENT1 in hyperthyroid atria. It is concluded that hyperthyroidism does not alter the prevailing direction of the Ado transport, moreover intensifies the Ado influx in the guinea pig atrium.